This invention provides the methodology and agents for treating any disease or clinical condition which is at least partly the result of endoplasmic reticulum-associated retention of proteins. Thus, the methods and agents of the present invention provide for the release of normally retained proteins from the endoplasmic reticulum. The present invention is particularly useful for treating any disease or clinical condition which is at least partly the result of endoplasmic reticulum-associated retention or degradation of mis-assembled or mis-folded proteins.
All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
A. Introduction
Protein folding and quality control machinery has been implicated in the molecular pathogenesis of several human diseases caused by defective intracellular transport of an aberrantly folded protein through the secretory pathway. Exemplary diseases include pulmonary emphysema resulting from severe plasma xcex1-antitrypsin deficiency and Cystic Fibrosis resulting from mutations in the cystic fibrosis transmembrane conductance regulator (Amara et al., Trends Cell. Biol. 2:145-149; Le et al., J. Biol. Chem. 269:7514-7519; Pind et al., J. Biol. Chem. 269:12784-12788). This invention is directed to the treatment and cure of such diseases.
Although the treatment and cure of Cystic Fibrosis and Chronic Obstructive Pulmonary Disease have been chosen as representative diseases for the purpose of describing and explaining the present invention, the compositions and/or methods of the present invention are applicable to the treatment and cure of any disease which involves the defective intracellular transport of mis-folded proteins.
B. Cystic Fibrosisxe2x80x94An Overview of the Disease, Protein and Gene
The Disease of Cystic Fibrosis
Cystic Fibrosis (CF) is an inherited multi-system metabolic disorder of the eccrine and exocrine gland function, usually developing during early childhood and affecting mainly the pancreas, respiratory system and sweat glands. Glands which are affected by CF produce abnormally viscous mucus, usually resulting in chronic respiratory infections, impaired pancreatic and digestive function, and abnormally concentrated sweat. CF is also called Clarke-Hadfield syndrome, fibrocystic disease of the pancreas and mucoviscidosis.
CF is the most common fatal autosomal recessive disease in Caucasians affecting approximately 1 in 2000 or 2500 live births, with 1 person in 25 being a heterozygote (Boat et al., Metabolic Basis of Inherited Disease 2649-2680 (McGraw-Hill, 1989)). It is a complex disorder mainly affecting the ability of epithelial cells in the airways, sweat glands, pancreas and other organs and tissues to secrete chloride ions (Clxe2x88x92), leading to a severe reduction of the accompanying sodium and water in the mucus. Thus, the primary defect in CF is thought to be the relative impermeability of the epithelial cell to chloride ions (Clxe2x88x92). This defect results in the accumulation of excessively thick, dehydrated and tenacious mucus in the airways, with subsequent bacterial infections, mucus blockage and inflammation. For a detailed discussion of the clinical manifestations, diagnosis, complications and treatment of the disease, see R. C. Bone, Cystic Fibrosis, In J. C. Bennett et al., Cecil Textbook of Medicine 419-422 (W. B. Saunders Co., 1996).
The CF Protein and Gene
The gene for CF is located on the long arm of chromosome 7. For a description of the gene, the expression of the gene as a functional protein, and confirmation that mutations of the gene are responsible for CF, see Gregory et al, Nature 347:382-386 (1990); Rich et al., Nature 347:358-363 (1990); and Watson et al., Recombinant DNA, pp. 525-529 (Scientific American Books, 1992).
The protein encoded by the CF-associated gene is the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a cyclic AMP-dependent chloride channel found in the plasma membrane of certain epithelial cells. CFTR contains approximately 1480 amino acids and is made up of two repeated elements, each comprising six transmembrane segments and a nucleotide binding domain. The two repeats are separated by a large, polar, so-called R-domain containing multiple potential phosphorylation sites. Based on its predicted domain structure, CFTR is a member of a class of related proteins which includes the multi-drug resistance or P-glycoprotein, bovine adenyl cyclase, the yeast STE6 protein as well as several bacterial amino acid transport proteins (Riordan et al., Science 245:1066-1073 (1989); Hyde et al., Nature 346:362-365 (1990)). Proteins in this group are characteristically involved in pumping molecules into or out of cells.
Gene Mutations Responsible for CF
The metabolic basis for CF results from a mutational defect in a specific chloride channel. Naturally-occurring, single amino acid mutations have been found in the first nucleotide binding fold of CFTR. Although over 800 different mutations have been identified in the CF associated gene, the most common is a deletion of three nucleotides which results in the loss of a phenylalanine residue at position 508 of CFTR (xcex94F508) (Davis et al., Am. J. Respir. Crit. Care Med. 154:1229-1256 (1996); Sheppard and Welsh, Physiol. Rev. 79:Suppl: S23-S45 (1999)).
Additional examples of CFTR mutants include G551D, a mutation in the CFTR gene resulting in a substitution of aspartic acid for glycine at amino acid 551 of the CFTR (U.S. Pat. No. 5,602,110), and several naturally-occurring CFTR mutants carrying a defect in the first nucleotide binding fold (NFB1) (U.S. Pat. No. 5,434,086).
Mutations at position 508 contribute to approximately 90% of all CF cases, although the percentage varies by race and geographical location (Kerem et al., Science 245:1073-1080 (1989)). This mutation results in the failure of an epithelial cell chloride channel to respond to cAMP (Frizzel et al., Science 233:558-560 (1986); Welsh, Scienc 232:1648-1650 (1986); Li et al., Science 244:1353-1356 (1989); Quinton, Clin. Chem. 35:726-730 (1989)). Although CF-affected epithelial cells are unable to normally up-regulate apical membrane Cl-secretion in response to agents which increase cAMP, they do increase Cl-secretion in response to increases in intracellular Ca2+.
There are at least three different chloride channels found in epithelial cells, including volume sensitive, calcium-dependent and cAMP-dependent. In normal individuals, chloride channels are located on the luminal membranes of epithelial cells. When these channels are open, chloride ions move into the airway lumen, producing an osmotic gradient that draws water into the lumen. In Cystic Fibrosis, the absence or dysfunction of at least one of these chloride channels, CFTR, results in the failure to secrete chloride in response to cAMP stimulation. Therefore, there is an inadequate amount of water on the luminal side of the epithelial membranes as well as excessive sodium reabsorption. In airway cells this causes abnormal mucus secretion with inadequate water content, ultimately leading to pulmonary infection and epithelial damage. Abnormal electrolytes in the sweat of CF patients probably results from the impermeability of the sweat duct epithelium to chloride.
Physiologically, the (xcex94F508) mutant CFTR is mis-folded and unable to assume its appropriate tertiary conformation (Thomas et al., J. Biol. Chem. 267:5727-5730 (1992)), is retained in the endoplasmic reticulum (ER) as a result of the mutation-induced mis-folding, and eventually is targeted for degradation (Cheng et al., Cell 63:827-834 (1990); Ward et al., Cell 83: 122-127 (1995)). Other examples of processing mutants leading to CFTR chloride channel dysfunction, with the frequency of the mutation in parentheses, include: D1507 (0.5), S549I (very rare), S549R (0.3), A559T (very rare) and N1303K (1.8) (Welsh et al., Cell 73:1251-1254 (1993)). P574H and A455E are additional CF-associated mutants which are also mis-processed (Ostedgaard et al., J. Cell. Sci. 112(Pt13):2091-2098 (1999)). Only 5% to 10% of the mis-folded CFTR protein of these two mutants reaches the apical membrane.
Because more than 98% of CF patients die from either respiratory failure or pulmonary complications before reaching maximum physiological maturity, the therapeutic goals have historically been to prevent and treat the complications of obstruction and infection in the airways, enhance mucous clearance, and improve nutrition. The identification of the xcex94F508 defect (and other mutations in CFTR) has facilitated the rapid development of proposed treatments for CF, including the therapeutic introduction of the wild-type CFTR gene via gene therapy, as well as more traditional drug therapies.
C. Current and Potential Treatments for Cystic Fibrosis
Treatment of Cystic Fibrosis Using Traditional Drugs
Traditional treatments for CF include chest physiotherapy (e.g., percussion and postural drainage), various broncodilators, nutritional supplements (e.g., pancreatic enzymes and vitamins), exercise and rehabilitation, and long-term oxygen therapy for chronic hypoxemia. Aerosolized amiloride has been administered to improve the quality of the secretions, thereby improving the air flow in CF patients (U.S. Pat. Nos. 4,501,729 and 4,866,072). Although these methods have increased the overall survival and physical comfort of CF patients, the traditional drugs and treatment methodologies do not cure the afflicted individuals and CF-afflicted persons often are not expected to live beyond their mid-twenties or early thirties. (R. C. Bone, supra).
DNase Treatment
One identified new drug treatment for CF has been the use of DNase, such as human DNase 1, which ameliorates one of the side effects caused by the defect in CFTR (New England Journal of Medicine 331:637-642 (1994)). Although the water content of bronchial secretions is probably the critical determinant of secretion viscosity, it is believed that DNA from lysed cells may add to this index.
Increased Permeability of Epithelial Cells to Clxe2x88x92
U.S. Pat. No. 5,384,128 discloses a method of treating CF which comprises administration of an epithelial cell chloride permeability enhancing composition which is a nontoxic, nonionic surfactant having (1) a critical micelle concentration of less than about 10 mM and a hydrophile-lipophile balance number of from about 10 to 20, and (2) a suitable hydrophobic organic group joined by a linkage to a suitable hydrophobic polyol. Examples of such compositions include a saccharide joined with organic groupings, such as an alkyl, aryl, aralkyl, or fatty acid group; polyoxyethylenes joined with an organic grouping; or, alkyl polyoxyethylene sorbitans. The preferred method of treatment is by aerosol inhalation.
Treatment of Cystic Fibrosis Using Gene Therapy
Several methods of gene therapy have been developed and are being tested for providing the normal CFTR gene into CF patients. For example, transfecting the normal CFTR gene into the nasal epithelial cells of patients has been shown to improve functions of the transmembrane chloride channel. These results have raised the hope that delivery of retroviral vectors containing normal CFTR genes directly to the lung epithelium by means of aerosol will help alleviate CF. Despite promising results, implementation of gene therapy methodologies to xe2x80x9ccurexe2x80x9d CF still remain in the experimental stages. As a result, an efficacious drug alternative to proposed gene therapy treatments is needed to more effectively treat CF.
D. Chronic Obstructive Pulmonary Disease: An Overview of the Diseases, Protein and Gene
The Disease
The designation Chronic Obstructive Pulmonary Disease (COPD) is an imperfect, although widely used, term because it includes several specific disorders with different clinical manifestations, pathologic findings, therapy requirements, and prognoses. The term encompasses chronic bronchitis and emphysema. Common to most of these diseases is chronic involvement of peripheral (small) airways or, more rarely, localized obstruction of central (large) airways. For a comprehensive overview of COPD, see Matthay et al., Chronic Airways Diseases, In Cecil Textbook of Medicine (Bennet et al., eds.; W. B. Saunders Company) 20th Ed., 52:381-309 (1996)).
Since elastase released by activated neutrophils is rendered inactive by the inhibitor xcex1-antitrypsin (AAT), diminished circulating levels of AAT can result in proteolytic destruction of lung elastin, a phenomenon implicated in the pathogenesis of COPD (Travis et al., Annu. Rev. Biochem. 52:655-709 (1983); Beith, Front. Matrix Biol. 6:1-4 (1978)).
The xcex1-Antitrypsin (AAT) Protein and Gene
Human AAT is a 394-amino acid protein glycosylated at three specific asparagine residues (Carrell et al., In Proteinase Inhibitors (Barrett et al., eds.; Elsevier, Amsterdam) 403-420 (1986); Long et al., Biochemistry 23:4828-4837 (1984); Yoshida et al., Arch. Biochem. Biophys. 195:591-595 (1979)). AAT is a member of the serine proteinase inhibitor superfamily (Huber et al., Biochemistry 28:8951-8966 (1989)). It is folded into a highly ordered tertiary structure containing three xcex2-sheets, nine xcex1 helices, and three internal salt bridges (Loebermann et al., J. Mol. Bio. 177:531-556 (1984)).
Gene Mutations Responsible for COPD
The human AAT structural gene is highly polymorphic and several alleles exhibit a distinct mutation predicted to preclude conformational maturation of the encoded polypeptide following biosynthesis (Brantly et al., Am. J. Med. 84:13-31 (1988); Stein et al., Nat. Struct. Biol. 2:96-113 (1995)). Genetic variants of human AAT unable to fold into the native structural conformation are poorly secreted from hepatocytes (Laurell et al., In Protease Inhibitors in Plasma (Putnam, ed.; Academic Press, New York) Vol. 1:229-264 (1975); Peters et al., In Plasma Protein Secretion by the Liver (Glaumann et al., eds.; Academic Press, New York) 1-5 (1983); Sifers et al., Semin. Liver Dis. 12:301-312 (1992); Sifers et al., In The Liver: Biology and Pathology (Arias et al., eds.; Raven Press Ltd., New York) 3rd Ed. 1357-1365 (1994)).
Choudhury et al. (J. Biol. Chem. 272(20):13446-13451 (1997)) report on a secretion-incompetent variant null of xcex1-antitrypsin designated as Hong Kong.
E. Overview of the Invention
The current invention is based on the unexpected discovery that inhibition of UGGT or other elements of the ER-chaperon retention machinery allows mis-folded or mis-assembled proteins, such as mis-folded mutant (xcex94F508) CFTR protein and mutant xcex1-antitrypsin (Hong Kong), to exit the ER instead of being targeted for degradation. By preventing the normal action of UGGT and/or other elements of the ER-chaperon retention machinery, the mis-folded proteins exit the ER and are targeted to the plasma membrane, where despite the mutation, they can function. This invention has practical applications in treating or curing any disorder or disease which directly or indirectly results from mis-folded ER proteins including, but not limited to, clinical conditions related to the misfolding and/or non-release of the transmembrane precursors of the glycosylphosphatidylinositol-linked proteins, low density lipoprotein receptor, the thyroid prohormone thyroglobulin (Tg), Class I histocompatibility proteins as occurs in tumors and in numerous viral infections, as well as CFTR and xcex1-antitrypsin.
While many groups are currently trying to overcome these types of diseases and clinical conditions through gene therapy, the approach of the present invention employs chemical pharmaceuticals to rescue the endogenous mutant protein. It is likely, therefore that our method will not be limited by the current challenges which confront gene therapy efforts, including low multiplicity of transformation, low levels of expression, and inflammation and immune responses to the requisite viral vectors. Recent deaths associated with experimental gene therapies further indicate the need for alternative treatment methods. Our approach is also the first to attempt to defeat ER retention of mis-folded proteins by interfering directly with ER quality control mechanisms.
As described in detail herein, this invention encompasses various compositions and methods which reduce the activity of any ER chaperone including, but not limited to, UGGT and thereby permit exiting of mis-folded and mis-assembled proteins from the ER. Such compositions include compounds which covalently bond to modified UGGT and irreversibly inhibit its catalytic function. Exposure to oligonucleotides whose sequences are antisense to the UGGT coding sequence will also reduce UGGT expression and activity. Optimal UGGT activity requires high concentrations of Ca2+. Our research also demonstrates that interfering with UGGT activity by depleting ER Ca2+ stores through various treatments, such as with calcium pump inhibitors, allows the mis-folded but functional xcex94F508 CFTR protein to xe2x80x9cescapexe2x80x9d from the ER and reach the cell surface. Thus, our discovery also provides novel and clinically applicable treatment for reversing or preventing diseases or clinical conditions which result from the ER-associated retention or degradation of mis-assembled or mis-folded glycoproteins.
This invention provides methods and reagents for treating any disease or clinical condition by administering an agent that permits the release of proteins from the ER. More particularly, this invention provides such methods wherein the disease or clinical condition is at least partly the result of endoplasmic reticulum-associated retention or degradation of mis-assembled or mis-folded proteins.
In one embodiment of the invention, methods are provided wherein the agent permits release of mis-assembled or mis-folded proteins from the endoplasmic reticulum. Preferably the mis-assembled or mis-folded proteins retain sufficient activity to relieve at least some of the symptoms of the disease or clinical condition.
In another embodiment of the invention, methods are provided wherein the proteins being released are glycoproteins.
The methods of the present invention are useful for treating diseases or clinical conditions such as Cystic Fibrosis, Chronic Obstructive Pulmonary Disease, Paroxysmal Nocturnal Hemoglobinuria, Familial Hypercholesterolemia, Tay-Sachs Disease, viral diseases, neoplastic diseases, Hereditary Myeloperoxidase Deficiency, Congenital Insulin Resistance, Rhinosinusitis, Hemochromatosis, Gitelman""s Syndrome, Cystinuria, and certain forms of Nephrogenic Diabetes Insipidus.
In one embodiment of the invention, the methods involve using agents which act as calcium pump inhibitors.
In another embodiment of the invention, the methods involve using agents which decrease or inhibit the functional activity of UDP glucose:glycoprotein glycosyl transferase.
In still another embodiment of the invention, the methods involve using agents that decrease or inhibit activity of the endoplasmic reticulum Ca++ ATPase.
In yet another embodiment of the invention, the methods involve using agents that lower the concentration of Ca++ in the endoplasmic reticulum.
In another embodiment of the invention, the methods involve using agents that cause release of Ca++ from the endoplasmic reticulum.
In yet another embodiment of the invention, the methods involve using agents that increase or stimulate IP3 receptor activity.
In yet another embodiment of the invention, the methods involve using agents that increase or stimulate ryanodine receptor activity.
In still another embodiment of the invention, the methods involve using agents that decrease or inhibit calnexin functional activity.
Examples of agents which are useful in the methods of the present invention include, but are not limited to, thapsigargin or a derivative thereof, cyclopiazonic acid or a derivative thereof, DBHQ or a derivative thereof, or halothane or a derivative thereof.
Additional examples of agents that are useful in the methods of the present invention include, but are not limited to, oligonucleotides which are antisense to UDP glucose:glycoprotein glycosyl transferase, calnexin or Ca++ ATPase.
The present invention also provides methods wherein the agents are administered to the pulmonary system, such as by using an aerosol.
The present invention provides methods of releasing a mis-assembled or mis-folded glycoprotein from the endoplasmic reticulum of a cell by administering an agent that decreases or inhibits the functional activity of UDP glucose:glycoprotein glycosyl transferase.
The present invention also provides methods of releasing a mis-assembled or mis-folded glycoprotein from the endoplasmic reticulum of a cell by administering an agent that decreases or inhibits activity of the endoplasmic reticulum Ca++ ATPase.
The present invention also provides methods of releasing a mis-assembled or mis-folded glycoprotein from the endoplasmic reticulum of a cell by administering an agent that lowers the concentration of Ca++ in the endoplasmic reticulum.
The present invention also provides methods of releasing a mis-assembled or mis-folded glycoprotein from the endoplasmic reticulum of a cell by administering an agent that decreases or inhibits calnexin functional activity.
The present invention also provides methods of increasing the permeability of the apical surfaces of airway epithelial cells to a chloride ion by administering an agent that decreases or inhibits the intracellular retention of mis-assembled or mis-folded glycoproteins.
The present invention further provides methods of increasing the permeability of the apical surfaces of airway epithelial cells to a chloride ion by administering an agent that decreases or inhibits the activity of UDP glucose:glycoprotein glycosyl transferase.
The present invention also provides methods of increasing the permeability of the apical surfaces of airway epithelial cells to a chloride ion by administering an agent that decreases or inhibits activity of the endoplasmic reticulum Ca++ ATPase.
The present invention further provides methods of increasing the permeability of the apical surfaces of airway epithelial cells to a chloride ion by administering an agent that lowers the concentration of Ca++ in the endoplasmic reticulum.
The present invention also provides methods of increasing the permeability of the apical surfaces of airway epithelial cells to a chloride ion by administering an agent that decreases or inhibits calnexin functional activity.
The present invention further provides methods of treating cystic fibrosis or alleviating the symptoms of cystic fibrosis by administering an agent that decreases or inhibits the activity of UDP glucose:glycoprotein glycosyl transferase.
The present invention also provides methods of treating cystic fibrosis or alleviating the symptoms of cystic fibrosis by administering an agent that decreases or inhibits activity of the endoplasmic reticulum Ca++ ATPase.
The present invention further provides methods of treating cystic fibrosis or alleviating the symptoms of cystic fibrosis by administering an agent that lowers the concentration of Ca++ in the endoplasmic reticulum.
The present invention further provides methods of treating cystic fibrosis or alleviating the symptoms of cystic fibrosis by administering an agent that decreases or inhibits calnexin functional activity.
The present invention provides methods of screening candidate compounds to identify an agent that inhibits endoplasmic reticulum-associated retention or degradation of a mis-assembled or mis-folded glycoprotein, wherein the method includes the steps of:
a) treating a cell exhibiting intracellular retention of a mis-assembled or mis-folded glycoprotein in the endoplasmic reticulum with the candidate compound; and
b) determining whether the mis-assembled or mis-folded glycoprotein is released from the endoplasmic reticulum, thereby identifying the candidate compound as an agent that causes the release of a malformed mis-folded glycoprotein from the endoplasmic reticulum.
The present invention also provides methods of screening candidate compounds to identify an agent that inhibits the functional activity of UDP glucose:glycoprotein glycosyl transferase, wherein the method includes the steps of:
a) treating a cell exhibiting intracellular retention of a mis-assembled or mis-folded glycoprotein in the endoplasmic reticulum with the candidate compound; and
b) determining whether the mis-assembled or mis-folded glycoprotein is released from the endoplasmic reticulum, thereby identifying the candidate compound as an agent that causes the release of a mis-assembled or mis-folded glycoprotein from the endoplasmic reticulum.
The present invention provides aerosol formulations of thapsigargin, DBHQ or cyclopiazonic acid.
In addition, the present invention provides compositions which include two or more of the following agents: 1) an agent that decreases or inhibits the activity of UIDP glucose:glycoprotein glycosyl transferase, 2) an agent that decreases or inhibits activity of the endoplasmic reticulum Ca++ ATPase, 3) an agent that increases or stimulates IP3 receptor activity, 4) an agent that increases or stimulates ryanodine receptor activity, and 5) an agent that decreases or inhibits calnexin functional activity.